Iridoid 1-O-glucosylation enzyme activities of crude cell-free extracts prepared from loganin-producing plants and nonproducing cultured cells were comparatively examined. Crude cell-free extracts from Lonicera japonica cell suspension cultures glucosylated 7-deoxyloganetic acid, 7-deoxyloganetin, and loganetin, but not iridotrial, an intermediate just preceding 7-deoxyloganetic acid. Crude cell-free extracts from Hydrangea macrophylla young leaves also glucosylated 7-deoxyloganetin and loganetin, whereas those from cultured cells induced from iridoid-nonproducing plants did not show any iridoid glucosylation activity. The partially purified glucosyltransferase from L. japonica cells showed the highest glucosylation activity for loganetin. However, kinetic studies showed that Km values for 7-deoxyloganetic acid, 7-deoxyloganetin, and loganetin were 106μM, 561μM, and 660μM, respectively, indicating that the enzyme had the highest affinity to 7-deoxyloganetic acid. These data suggest the presence of a pathway for the biosynthesis of loganin, in which 7-deoxyloganetic acid is glucosylated at the 1-O position to give 7-deoxyloganic acid, which is further hydroxylated and methylated to produce loganin.
Sedum drymarioides, an endangered plant, was micro-propagated, and the regenerated plants showed the CO2 exchange pattern of a C3-plant. Stem and leaf explants were cultured on solid MS medium containing various concentrations of 1-naphtaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), resulting in callus formation at 0.1-1mgl-1 NAA. From the stem callus, the best differentiation of root and bud was observed at 1mgl-1 of NAA plus 0.1mgl-1 of BAP and 0.1mgl-1 of NAA plus 1 mgl-1 of BAP, respectively. From the leaf callus, the differentiation was most efficient with the combination of 1mgl-1 of NAA plus 1mgl-1 of BAP and 1mgl-1 of NAA plus 10mgl-1 of BAP, respectively. The buds further multiplied on medium containing 0.1mgl-1 of NAA and 10mgl-1 of BAP. They developed into rosette plantlets and grew to normal size on phytohormone-free MS medium in glass bottles, where they bolted and flowered. The regenerated plantlets were successfully transferred to pots where fertile seeds were obtained.
Under the control of Arabidopsis triptophan synthase β subunit promoter (PTSB1), the oat thionin gene (Asthi1) was introduced into carnation (Dianthus caryphyllus L.) via Agrobacterium-mediated gene transfer. Leaf bases of three cultivars of carnation (‘Scania’, ‘Percian Pink-Sim’ and ‘U. Conn. White Sim’) were infected with Agrobacterium harboring the binary vector with PTSB1::Asthi1 and hygromycin genes, and cultured on the medium containing hygromycin. Independent 99 lines resistant to hygromycin were regenerated and the introduction of the transgene was confirmed in 11 lines by polymerase chain reaction. Transgenic carnation plants exhibited the resistance against infection with Burkholderia caryophylli which caused bacterial wilt, and the levels of resistance correlated with those of the thionin gene transcript.
To develop a transformation system for pear cultivars (Pyrus communis L.), we investigated the ability of cotyledon explants produced from seeds of mature fruit to regenerate, and monitored transgene expression during the early stages of the transformation procedure. The greatest shoot regeneration was induced on MS medium, supplemented with 2μM NAA and 30μM TDZ for ‘La France’ cotyledons (40%), and supplemented with 8μM NAA and 30μM TDZ for ‘Bartlett’ cotyledons (75%). Of the three cytokinins (TDZ, BA and Zeatin) examined, TDZ was the most effective for adventitious bud formation. To investigate the transfer of genes into pear cotyledon explants, we used a modified GUS gene that is expressed in plant tissues exclusively. Gene transfer was measured via transient expression of the GUS gene. The greatest frequency of gene transfer (82.7%) occurred when the explants were co-cultivated in the dark for 7 days after being inoculated with Agrobacterium tumefaciens strain EHA101 without pre-culture. A practical transformation was also conducted, in which carnation cDNA encoding an ACC oxidase (DC-ACO) was induced into cotyledon explants of two pear cultivars. Four transgenic ‘Bartlett’ seedlings and two ‘La France’ seedlings were obtained. Simultaneously, three lines of nontransformed control plant that had the same genome as the transgenic plants were obtained by inducing regeneration of a noninoculated cotyledon derived from the same seed as an inoculated cotyledon. PCR and Southern blot analyses confirmed the integration of the transgene in the genomes of all the transgenic lines.
We used flow cytometry to analyze chromosomal changes in regenerants of the Kalanchoe blossfeldiana cultivar ‘Tetra Vulcan’ (4x). About 80% of regenerants showed increased ploidy levels. Twenty-four (20.7%), 87 (75.0%), 1 (0.9%), and 4 (3.4%) regenerants had ploidy levels of 4x, 8x, 12x, and 16x, respectively. 4x and 8x regenerants grew normally and similarly to wild-type plants, but 12x and 16x plants showed remarkable delays in growth. Plant height and leaf size among the 4x and 8x plants were the same as in wild-type plants, but those of the 12x and 16x plants were dramatically reduced. Leaves of plants with ploidy levels of 8x or more were thicker than those of 4x plants, and this character increased with increasing ploidy level. Our results confirmed that regeneration from leaf segments is an efficient method of polyploidization in Kalanchoe.
We succeeded in establishing a stable and efficient transformation system of chrysanthemum (cv. Shuho-no-chikara) which could eliminate both the appearance of the chimeric regenerants consisting of transgenic and non-transgenic tissues and that of the transgene inactivated regenerants. We compared two transformation systems, callus induction (CI) system and adventitious shoot induction (SI) system. The transformation frequency in CI system (4.4%) were higher than that in SI system (0.3%). All regenerated plantlets obtained by CI system express gus gene stably even after vegetative propagation. While a few regenerants obtained by SI system have gus gene and express gus gene in chimeric manner. Then we applied the CI system in other famous and commercial cultivars of chrysanthemum and obtained transformed plants with high transformation frequency in 15 among 21 cultivars. Regarding the stable gus gene expression in all regenerants, CI system should eliminate gene inactivation in regenerants and is beneficial for the production of genetically engineered chrysanthemum.
Somatic hybrids were produced between embryogenic callus protoplasts of ‘Shogun’ mandarin (Citrus reticulata Blanco, 2n = 2x = 18) and leaf protoplasts of grapefruit (C. paradisi Macf., 2n = 2x =18) by electrofusion. Hybridity of the two regenerated plants was confirmed by leaf characteristics and nuclear genome analysis using RAPD and PCR-RFLP. Flow cytometry and root-tip cell count of the two hybrids revealed a chromosome number of 27. These triploid somatic hybrids on transfer to field showed normal morphology and vigorous growth. Field performances as well as disease resistance are under investigation.
Storage organ of Dioscorea opposita was efficiently generated by using a drum type bioreactor. This reactor was not equipped with a system for mechanical aeration and explants were immersed intermittently into the medium on rotation. When explants precultured on a rotary shaker were transferred to the bioreactor, approx. 230 pieces of storable organs that could be bulbils or microtubers were generated after 5 weeks. These organs sprouted within a week and eventually grew intonormal plants.
Shoot-generating tissue cultures of Oenothera tetraptera that produce polyphenolic compounds were established. The tissues, which were subcultured on LS agar medium containing 10μM IAA and 10μM kinetin, produced galloylglucoses and monomeric and dimeric ellagitannins. Plants were regenerated from the tissues upon transfer from the growth regulator-containing medium to hormone-free medium. Plant regeneration from Oenothera shoots was accompanied by changes in the composition of hydrolysable tannins (galloylglucoses to oligomeric ellagitannins).
Transgenic plants of Oenanthe javanica (Blume) DC. ‘Shimane-Midori’ were obtained by Agrobacterium-mediated gene transfer. Embryogenic calli derived from petiole segments were infected with Agrobacterium tumefaciens strain EHA101, harboring a binary vector pIG121Hm that included genes for GUS, kanamycin resistance and hygromycin resistance. After four days of co-culture on MS medium containing 0.1mgl-1 2, 4-D, the infected calli were transferred onto hormone-free MS medium containing 500mgl-1 carbenicilin. Seven days after the start of hormone-free MS medium, the calli were transferred onto MS medium containing 50mgl-1 hygromycin for selection. Approximately four month after infection, seventy-one hygromycin-resistant plantlets were formed from about 4g calli. Expression of the GUS gene was confirmed by histochemical assay. The results of Southern hybridization analysis indicated that at least three independent transgenic plants were obtained, and all of them had a single copy of the GUS gene in their genomes.
Sixteen colored cell lines of Portulaca sp. ‘Jewel’ forming betalain were established, seven cultured on modified Murashige and Skoog (mMS) medium and nine on modified Girod and Zrÿd (mJ1). Each line was different in term of betalain content. Both betacyanin and betaxanthin widely varied in quality and quantity from line to line. A magenta-colored phenotype contained the maximum betacyanin and an orange-colored phenotype contained the maximum betaxanthin. Modified MS medium was optimal for betacyanin synthesis whereas modified J1 medium synthesized more betaxanthin. Betacyanin and betaxanthin both accumulated in a positive correlation to the cell growth in our culture system, and the highest contents were recorded during the logarithmic phase. The addition of natural auxin, indole-3-acetic acid (IAA), to both types of culture media instead of synthetic auxin, 2, 4-dichloro-phenoxyacetic acid (2, 4-D) inhibited the growth and reduced betalain synthesis. But a few subcultures after transfer to IAA-containing medium, two cell lines were established one producing betacyanin and one betaxanthin. Different cell phenotypes (either magenta or yellow colored cells) exhibit same responses in their requirement of light. Cellular betacyanin and betaxanthin drastically increased under continuous illumination, particularly with blue light irradiation along with the increasing number of growth cycles.
A successful transformation method was reported on Delphinium spp., using Agrobacterium tumefaciens. Elongated hypocotyl and cotyledon petiole segments were infected with Agrobacterium tumefaciens strain LBA4404, which harbored a binary vector plasmid, pIG121-Hm, which included the β-glucuronidase (GUS) gene (with an intron) as a reporter gene, and the neomycin phosphotransferase II gene and the hygromycin phosphotransferase gene as selection markers. Explants were cultured on Murashige and Skoog medium supplemented with 1.0mgl-1 thidiazuron, 1.0mgl-1 2, 4-dichlorophenoxyacetic acid, 300mgl-1 ticarcillin, and 5mgl-1 hygromycin or 100mgl-1 kanamycin (selection medium) for regeneration. Transformation was confirmed by histochemical assays of GUS activity in plant tissues, and by PCR analysis of the GUS gene. Through four experiments, six independent GUS-positive regenerants were obtained out of 1276 explants.
Mass propagation of the Japanese angelica tree (Aralia elata seem.)‘Zaoh’ line No.2 was established through somatic embryos. Petioles of leaflets were cultured for induction of calli on an MS medium containing 1mgl-1 of 2, 4-D in combination with 0.5mgl-1 BA. The initiated calli were moved onto MS medium supplemented with glutamine 450mgl-1 and asparagine 300mgl-1. An embryogenic callus developed after 5months and was moved onto regulator-free MS medium. Numerous plantlets were regenerated from this embryogenetic callus, and rooted plantlets were potted after acclimation and planted to the field. The plants had the same characteristics in morphology in the field, except for the number of thorns per internode. The plants also had the same resistance to Phytophthora disease as was in the original genotype ‘Zaoh’ line No.2. This study provided a method for mass propagation from petioles of leaflets and proves that regenerated plants maintain the morphogenetic characteristics and disease resistance of the original genotype.
Interspecific somatic hybrids were obtained by electric protoplast fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. chmielewskii (LA1330). A multiple-step selection procedure was used in selecting the hybrids depending on IOA treatments and a culture medium that only allows cotyledon protoplasts to regenerate. Plants were regenerated on the calli derived from fused protoplasts. After acclimation, the hybrid plants with fruits through self-pollination were obtained. Identification of the regenerated putative hybrid plants was carried out by morphological, chromosomal and random amplified polymorphic DNA (RAPD) analysis, as well as restriction fragment length polymorphism (RFLP) comparison of parental and hybrid plants. The hybrids showed unique morphologies in leaf, flower and stem compared with that of two fusion partners, 2n = 4x = 48, 47, 69-72 chromosomes, both of the parental-specific RAPD bands, and genomic recombination and elimination occurred in mitochondria level. Cell fusion technique shows its new potential in foreign gene transformation between L. esculentum and L. chmielewskii.
For the metabolic engineering of bio-functional substances in Citrus, metabolic changes of limonoids were investigated in embryogenic cell cultures of Citrus and Fortunella. When embryogenic cells of Citrus and Fortunella were cultured for four weeks in the liquid subculture medium containing nomilin, most species converted nomilin to obacunone and limonin. In addition, calamin group limonoids were produced in Fortunella and Calamondin cells. Deacetylnomilin was produced from nomilin in the cells of C. ichangensis and its related species. Exogenously added limonin into medium was not metabolized. The embryoid formation in C.aurantium did not affect the limonoid metabolism. All cells cultured without the presence of nomilin or limonin did not generate any limonoids. Furthermore, transgenic C. sinensis cell harboring a chimeric limonoid UDP-glucosyltransferase (limonoid GTase) gene was generated. The conversion of limonin into limonin 17-β-D-glucopyranoside (LG) demonstrated an efficient assay system for the transgene.